The present invention relates to a sugar chain purification instrument for purification by separation of sugar chain from glycoprotein.
For analysis of sugar chain structure of glycoprotein, it is necessary to separate the sugar chain from glycoprotein. The separation is carried out either by liberating the sugar chain with enzyme or by chemically breaking glycosidic linkage.
As the enzymes which liberate asparagine-linked sugar chain, there are glycopeptidase derived from almonds (Biochem. Biophys. Res. Commun., 76, 1194), glycopeptidase derived from sword beans (Biochem. Biophys. Res. Commun., 112, 155), and N-glycanase (J. Biol. Chem., 259, 10700). Furthermore, as a method for carrying out chemical breakage between sugar-protein, hydrazinolysis is mostly utilized, and other methods for chemical breakage of sugar chain include trifluoroacetolysis and use of LiBH4.
When comparison is made on merits and demerits of the enzyme method and chemical method for breakage of sugar chain of glycoprotein, the merit of the enzyme method is that the operation of this method is considerably simpler than the hydrazinolysis. That is, the enzyme method does not require complete drying of samples and requires only leaving the sample overnight with addition of enzyme. Post-treatment of the reaction mixture is also simple, and the sugar portion is fractionated by carrying out a gel filtration using a suitable column. Another merit of the enzyme method is that no modification occurs on the broken sugar chains.
In the hydrazinolysis, acetyl group of N-acetylhexosamine is eliminated, and hence hexosamine must be again N-acetylated. Furthermore, in glycoprotein derived from vegetables, fucose is linked through α-1,3 linkage to N-acetylglucosamine linking to aspargine, and it is reported that this fucose is readily liberated especially under the conditions of hydrazinolysis (Glycoconjyugate J., 3, 27).
Furthermore, when an enzyme is allowed to directly act on glycoprotein, sugar chain of the glycoprotein may not completely be liberated due to the steric hindrance caused by the higher order structure of protein, and, on the other hand, in the hydrazinolysis, the protein to which the sugar chain links is decomposed to amino acid, and hence the hydrazinolysis is not effective to presume the higher order structure of glycoprotein. It is known that the glycopeptidase derived from almonds which is most frequently used at present acts on any sugar chains without discrimination of structure among aspargine-linked sugar chains which are asialated, and furthermore an especially high reaction efficiency is exhibited in case the glycoprotein is decomposed to glycopeptide molecules which are made smaller to some extent by peptidase treatment or the like. However, the hydrazinolysis is superior in that aspargine-linked sugar chain can be cut out irrespective of presence or absence of sialic acid in the sugar chain or irrespective of the higher order structure of protein.
From the above, it can be considered that purification of sugar chain of glycoprotein by two-stage enzyme reactions which include digestion of protein portion with peptidase as a former stage before reaction with glycopeptidase which can perform moderate and simple liberation of sugar chain is very effective for analysis of structure of sugar chain of glycoprotein. With progress of microbiology and biochemistry, biocatalysts such as microorganisms and enzymes are utilized widely and industrially, but in many cases, the process is batch-wise, which comprises introducing a biocatalyst into a substrate solution and removing the biocatalyst after completion of reaction.
One example of conventional process is shown in the right column of the table shown in FIG. 1.
According to the conventionally employed batch process, a large reaction vessel must be used for every batch and hence a large plant is required. Furthermore, since it is very difficult to recover and reuse the biocatalyst which has been once used, the biocatalyst which has been used for one reaction is abandoned. Enzymes are originally extracted from organisms, and are expensive even in a small amount. Therefore, the conventional processes which abandon the enzymes used for one reaction are considerably uneconomical.
The object of the present invention is to provide a sugar chain purification instrument which can continuously and efficiently carry out purification of sugar chain.